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    Home > Biochemistry News > Biotechnology News > Selection and preparation of thin layer chromatography solvent/developing agent/color developer and matters needing attention

    Selection and preparation of thin layer chromatography solvent/developing agent/color developer and matters needing attention

    • Last Update: 2020-10-25
    • Source: Internet
    • Author: User
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    related topics
    thin layer analysis (T LC) technology
    thin layerchromatography method summary
    1. Method principle
    (1) flow phase using capillary force with the sample through the fixed phase.
    (2) The interaction between the sample and the fixed phase refers to the separation of the components during the transfer process due to the interaction of the even pole - (induced) - the even pole, the
    hydrogen bond and the role of van der Worrh, and other separations such as delay, adsorption, dispersion, ion exchange and complex.
    2. Solvents
    the solvent used must be "analytically pure" or "chromatography pure" and the solvent composition consists of a volume ratio (e.g. orthotinol - ice acetic acid - water - 4:1:1, V/V/V), or an absolute amount (e.g. 18 ml toluene and 2 ml methanol). The total amount should be sufficient immersion the TLC/HPTLC board at a depth of approximately 5mm. The expander requires fresh preparation, do not use repeatedly many times, if layering is required, then place the layered after taking the required phase (upper or lower layer), spare.
    , solvent selection rules:
    1, consider the polarity of the separation components, solubility, adsorption.
    2, first add a small polar solvent, if not to add a small amount of polar large solvent
    3, generally according to the principle of similar phase solubility, it is necessary to note that the polarity difference is large insoluble.
    4, mixed solvents usually use a high polarity and low-grade solvent composed of mixed solvents.
    5, the proportion of expanders depends on trying. Generally according to the literature reported in this type of compound with what kind of expander, first try to use this type of expander, and then keep trying the scale until a good separation effect of the expander is found.
    6, generally mixed with two solvents, the use of high polarity / low polarity volume ratio of 1/3 of the mixed solvent, if there are signs of separation, and then adjust the proportion (or add a third solvent) to achieve the best results;
    , the selection conditions of the expander:
    (1) have good solubility of the desired ingredients;
    (2) the RF of the part to be measured is between 0.2 and 0.8, and the quantitative determination is between 0.3 and 0.5;
    (3) does not respond to thebiopics with the parts to be tested or adsorbents; (5) the boiling point is moderate and the viscosity is small;
    (6) after expanding the speckle circle and concentration; (7) mixed solvent is best made with fresh preparation.
    , solvent polarity parameter table
    cyclane: -0.2, petroleum ether (class I. class, 30 to 60 degrees C), petroleum ether (class II. class, 60 to 90 degrees C), positiveane: 0.0. 0, toluene: 2.4, xylene: 2.5, benzene: 2.7, dichloromethane: 3.1, isopropyl alcohol: 3.9, butanol: 3.9, tetrahydrofuran: 4.1 0, chloroform: 4.1, ethanol: 4.3, ethyl acetate: 4.4, methanol: 5.1, acetone: 5.1, acetylene: 5.8, acetic acid: 6.0, water: 1 0.2
    1, generally speaking, the basic two phases of the weak polar solvent system consist of positive hexane and water, and then add methanol, ethanol, ethyl acetate as needed to regulate the polarity of the solvent system, in order to achieve a good separation effect, suitable for the separation of alkaloids, flavonoids, terpenes, etc.
    2, medium-polar solvent system consists of chloroform and water basic two phases, by methanol, ethanol, ethyl acetate, etc. to adjust, suitable for terpenes, coumerin, as well as some of the more polar lilac and steroid separation;
    3, strong polar solvent, composed of orthodol and water, but also by methanol, ethanol, ethyl acetate, etc. to adjust, suitable for the separation of very polar alkaloids.
    , the choice of
    agent molecule chemical structure, usually by the more polar part and non-polar part of the two parts. For example, with phenyl propane as a small part of polarity, with the increase of polar group part, the overall polarity increased, the polarity of the expander also increased.
    the polarity of different compounds and their corresponding expanders are discussed separately from the following.
    1, less polar volatile substances
    ice sheet: petroleum ether (30 to 60 degrees C) - ethyl acetate (17:3), thick phenol: phenyl-acetic ethylate (9:1.5),
    and alpha; alpha; - vancodone: benzene-acetate Ethyl-ice acetic acid (92:5:5), danpiphenol: ethyl cyclohexane-acetic acid (3:1),
    Conclusion: Petroleum ether, positive alkane and benzene as larger solvents, usually the role of dissolved and isolated compounds, and ethyl acetate as a solvent to regulate rf (ratio shift). In order to reduce the effects other than other similar solubility principles such as tailings, additives such asorganicor organic alkalis.
    2, less volatile substances with less polarity
    and beta; - Glutasterol: ethyl acetate-methanol (6:2.5:1) or cyclohexane-acetone (5:2),
    bear fruitic acid: toluene-acetate-ice acetic acid (5:2) 12:4:0.5), Chia fruit acid: chloroform-methanol (40:1),
    pig deoxycholic acid: chloroform-ether-ice acetic acid (2:2:1),
    Davine: benzene-ethyl acetate-methanol (15:2:0.2) or benzene-ethanol (8:1),
    dansanone II.A: benzene-acetate-formic acid (40:25:4), perforated lily esters: chloroform - Waterless ethanol (9:1)
    indigo red, indigo chloroform-ethanol (9:1) or benzene-chloroform-acetone (5:4:1)
    conclusion: the propulsion of these substances is more extreme than the less polar volatile substances, Because the mother nuclei of these substances are small, and the polar groups can usually form hydrogen bonds, such as carp acid, hydroxyl. The above substances, the decreased molecular weight of the parent nuclear, the increase of unsaturated health in the maternal nuclear structure (especially the appearance of benzene rings), the increase of polar groups, all make the polarity increase, the polarity of the expander also increased. There are many substances in this range, and the solvents of the general expander can be selected according to the polarity requirements from the order of cyclohexane-toluene-xylene-benzene-chloroform. It is noted here that isopropanol, orbutanol polar index is also relatively small, in this range of compounds are rarely used, because the viscosity is large, slow expansion, resulting in speckle diffusion; Solvents that regulate rf values, from ethyl acetate-methanol-acetone-ethanol. Volatile substances also have a lot of carbide, hydroxyl, but from its volatility can be understood, the inter-molecular force is not strong, in addition, the structure difference between the parent core and petroleum ether, positive alkanes and benzene is small, estimated to be easier to escape from silicone adsorption, faster into the solvent, without the need to improve the polarity of the expander.
    the structure of the glycosides is smaller due to the presence of polyhydroxys of sugar. Large organic solvents (chloroform, ethyl acetate, methanol, orthodol) and water are used in the expander. The use of acetic acid and foric acid, on the one hand, increases the polarity of the expander, but also can inhibit the role of silicone hydroxyl, reduce tailings. The use of water and acids is limited due to the limitations of solubility and silicone acid resistance.
    3, the polar smnut molecule organic acid:
    no food acid: chloroform-acetate ethyl acetate-formic acid (5:4:1), awei acid, coffee acid, chrysanthemum acid, chlorogenic acid, isogreen acid. For example, with phenyl propane as a small part of polarity, with the increase of polar group part, the overall polarity increased, the polarity of the expander also increased. In order, cinnamon acid, awei acid, coffee acid, chrysanthemum acid, chlorogenic acid. The corresponding expanders are: positive hexane-ether-ice acetic acid (5:5:0.1), benzene-ice acetic acid-methanol (30:1:3), chloroform-methanol-methiciltic acid (3 9:1: 0.5), petroleum ether-ethyl acetate (3:6:1), butyl acetate-foric acid-water (7:2.5:2.5).
    conclusion:
    Most of these substances are styrene nuclei, the polarity of the structure itself is relatively large, in addition to phenolic hydroxyl and carboxyl acid groups, individual polyhydroxybicides. The expander of saponin is similar and polar. Note that foric acid usually refers to a concentration of about 85% and contains water.
    4, nitrogen-containing organic matter:
    hydrochloric acid small pyridine: benzene-acetate-methanol-isopropyl alcohol-enriched ammonia test fluid (12:6:3:3:0.6) (ammonia vapor saturation) or ordol-ice acetic acid-water (7:1:2),
    Ephedrine: chloroform-methanol-enriched ammonia test fluid (20:5:0.5) or butanol-ice acetic acid-water (8:2:1)
    ammonium glyphosate: ethyl acetate-formate-ice acetic acid-water (15:1:1:2).
    conclusion analysis: Because the role of NH2 siliconol base is very strong, in the strong polar expander with organic acid, organic alkali sweep tail. For polar compounds, the use of butanol has less effect on speckle diffusion because of the strong role of compounds and silicones.
    when the separated substance is a weak polar substance, the weak polar solvent is generally chosen as an emphot agent, and when the separated substance is a strong polar component, the polar solvent must be selected as an ebbing agent. If a less adsorptive adsorption agent (e.g. silicone is replaced with diatom soil or talcum powder) for a particular polar substance, the polarity of the wasterion agent must also be reduced accordingly.
    3.TLC's universal color-showing method
    ideal color-showing hope high sensitivity, stable speckle color, good contrast between spots and background, speckle size and color depth is directly related to the amount of matter, In the case that the composition of the sample is not fully known, the universal color method is particularly important, the general color method is mainly:
    E/m T2K gV" p Fg4244131, ultraviolet treatment method: convenient, do not destroy the sample;
    Pl o; E r"m-z s? d T$V4244132, iodine vapor method: strong versatility, combined sensitivity with ultraviolet law is higher than the two methods used alone;
    $b 8M"? (P.e. e4244133, fluorescence reagents: the manufacture of fluorescent background, so that the original ultraviolet fluorescent-free substances are identified, fluorescent substances are more obvious; small woodworm academic blog JM0D6 (Rf;r
    4, sulfate solvent: effective but destructive for the vast majority of organic matter.
    4. Thin layer analysis and paper chromatography operation notes
    1, dot-like sampler sample on the thin sheet, generally do not affect the detection of dots, dot-like baseline from the bottom edge 1.0 to 1.5cm, sample diameter is generally not greater than 2mm, the distance between points can be seen speckle diffusion situation to not affect detection. If the sample solution is too thin, can repeat the sample, but should wait until the previous point of the solvent volatile before re-ordering, in order to prevent the sample point is too large, resulting in tailing, diffusion and other phenomena, and affect the separation effect. Dot sample must pay attention not to damage the thin layer surface
    2, expand the thin sheet of the sample into the expander of the expander, immersion in the depth of the expander 5mm from the origin is appropriate, sealed, to be expanded to the specified distance (generally 8 to 15cm), take out the thin sheet, dry, to be tested.
    : If the expander needs to be pre-balanced with the expander, the appropriate amount of expander can be added to the cylinder, closed, generally maintained for 15-30 minutes.
    3, color display and inspection test products containing colored ingredients in visible light can be viewed directly in daylight, but also can be used by spray or impregnation method to appropriate color display agent, or heating color, in daylight. Fluorescent substances or substances that excite fluorescence in certain reagents can be observed in 356nm UV lamps. For colorless visible light, but absorbed in ultraviolet light, silicone plates with fluorescent agents (e.g. GF254 plates) may be used to observe the chromatography of fluorescent annihilation material on the fluorescent plate surface under 254nm UV light.
    'll tell you my ancestral secret recipe
    . PE (60-90) EtAc/PE:1:2 EtAc/PE/AcOH=15:5:1 EtAc/AcOH/n-Butanol/H2O=2:1:1:1
    8 years I have used these four systems without any problems. I've been using ethyl acetate: cyclane, constantly adjusting the ratio until there is a satisfactory RF value, there is a tail may have to add acid or alkali, I generally acetic acid and triethylamine.
    I've been using it for years. Give it a try!
    The uniform slurry used for the
    -paved plate should not be too thick or too thin: too thick, the plate is prone to drag or pause caused by the layer pattern; The ratio of slurry is generally silicone G: water: 1:2 to 3, silicone G: sodium methyl cellulose solution, 1:2. The time of grinding slurry, according to experience to determine, and air humidity, generally by picking up the research rod when the situation of the slurry drop to judge, the thicker the more difficult the drop. In addition to affecting the smoothness of the plate, the thinning of the slurry also affects the thickness of the plate coating and further affects the amount of sample. The coating is thin and easy to overload, and the coating is thick and the color is not obvious. In general, the quality of the plate on the thin layer identification of the impact is not very large, shadow.
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